1. Field of the Invention
The present invention relates to human antithrombin III (AT III) mutants which are obtained by mutating one or more amino acid(s) in the amino acid sequence of human AT III into another amino acid(s) and exhibit high antiprotease activities even in the absence of heparin. These human AT III mutants are usable as a remedy for thrombotic disorders.
2. Description of the Related Art
Anticoagulant activity of glycosaminoglycans, including heparin, is mediated by antithrombin III (AT III) and heparin colactor II (HC II) contained in the blood. AT III and HC II are serlne protease inhibitors which are called serpins in general. There often has been reported, with respect to AT III, among these substances, that a decrease in the blood AT III level due to a congenital or acquired factor would result in thrombotic disorders. Accordingly, AT III plays a physiologically important role as a factor regulating the blood coagulation system consisting of a series of serine proteases.
It is known that human AT III is a glycoprotein of a molecular weight of approximately 60 kd which is mainly synthesized in the liver and contained in normal plasma at a concentration of about 150 .mu.g/ml and that human AT III inhibits serine proteases participating in coagulation and fibrinolysis systems including thrombin and factor Xa. The primary structure of human AT III has been clarified by the direct determination of its amino acid sequence (see Petersen, T. E. et al., The Physiological Inhibitors of Blood Coagulation and Fibrinolysis, Elsevier Science Publishers, Amsterdam, 43, 1979) and cDNA cloning [see Bock, S. C. et al., Nucl. Acids Res., 10, 8113 (1982); Prochownik, E. V. et al., J. Biol. Chem., 258, 8389 (1982); Chandra, T. et al., Proc. Natl. Acad. Sci. USA, 80, 1845 (1983)]. According to these reports, human AT III is a single-chain glycoprotein consisting of 432 amino acids which is secreted and formed by excising a signal peptide of 32 residues from a precursor protein. It has four N-linked glycosylation sites in the molecule. The carbohydrate content is about 15% of the molecular weight.
Human AT III reacts with a serine protease such as thrombin at a ratio of 1:1 and thus forms a stable complex, thus inhibiting the activity of the protease. It is thought that, in this reaction, a peptide bond between the 393rd Arg residue and the 394th Set residue in the molecule of human AT III is cleaved by the protease and an acyl bond is formed between the terminal Arg residue newly formed and the Ser residue at the active center of the protease. This Arg (393)-Ser (394) sequence is generally referred to as a reactive site.
The protease inhibition by AT III would relatively slowly proceed. When the reaction system contains heparin, however, the reaction is dramatically accelerated. Namely, the addition of heparin elevates the thrombin inhibition rate of AT III by more than 1,000 times. It is thought that this function mechanism proceeds as follows. When heparin binds to a specified site (heparin binding site) in AT III, the higher-order structure of AT III turns into a structure liable to undergo interaction with the protease. At the same time, tile protease binds to the heparin molecule. Thus a ternary complex is apt to be formed. Further, from the physiological viewpoint, it is considered that heparin-like substances existing on the surface of vascular endothelial cells exert similar actions and thus play an important role in the mechanism for regulating the blood coagulation system by AT III.
There have been used so-called anticoagulants for treating and preventing thrombotic disorders induced by various causes. Heparin is one of highly important anticoagulants at present. However, it is reported that serious side effects are sometimes induced by the administration of heparin [see Amerena, J. et al., Adverse Drug React. Acute Poisoning Rev., 9, 1 (1990); Levine, M. N. et al., Semi. in Thrombos. Hemostas., 12, 39 (1986); Kelton, J. G. et al., ibid., 12, 59 (1986) Levine, M. N., ibid., 12, 63 (1986)]. Typical examples of these side effects include hemorrhage, thrombocytopenia, hypoadrenalism, hypersensitiveness, necrosis of the administration site and osteoporosis. When there is a high risk of hemorrhage in the fields of, for example, obstetrics and gynecology or postoperative treatments or in the case of a prolonged administration, heparin should be carefully used. Furthermore, it is reported that heparin promotes inactivation of AT III by elastase of neutrophils in vitro [see Jordan, R. E. et al, Science, 237, 777 (1987); Jordan, R. E. et al., J. Biol. Chem., 264, 10493 (1989 )]. Thus care should be taken in the administration of heparin when elastase of neutrophils seemingly relates to the conditions of diseases such as serious infection or septicemia. In addition, the anticoagulant effect of heparin is essentially mediated by AT III and, therefore, can be scarcely expected in the case where blood AT III level is lowered.
Meanwhile, human AT III has been clinically applied to thrombophilia based on congenital AT III deficiency and disseminated intravascular coagulation syndrome (DIC) accompanied by a decrease in AT III in the form of a plasma derived AT III concentrate. As described above, however, AT III exhibits only a slow progressive antithrombin activity in the absence of heparin. Therefore the use of AT III alone is rather a supplementary treatment and its usefulness as an anticoagulant is limited. Thus attempts have been made to use AT III together with heparin or to prepare and use an AT III/heparin complex to thereby improve the usefulness of AT III as an anticoagulant. However, it is obvious that the above-mentioned disadvantages of heparin cannot be overcome even by these methods.
As described above, AT III has two functional sites, namely, the reactive site and the heparin-binding site. A number of reports have revealed that the amino acid sequence around the reactive site carries an important role in the expression of the function as a protease inhibitor as well as in the determination of inhibition specificity against various proteases. In congenital AT III anomalies such as AT III Hamilton wherein Ala at the 382-position has mutated into Thr [see DevraJ-Kizuk, R. et al., Blood, 72, 1518 (1988)], AT III Cambridge I wherein Ala at the 384-position has mutated into Pro [see Perry, P. J. et al., FEBS Lett., 254, 174 (1989)], AT III Glasgow wherein Arg at the 393-position has mutated into His [see Erdjument, H. et al., J. Biol. Chem., 263, 5589 (1988)], AT III Pescara wherein Arg at the 393-position has mutated into Pro [see Lane, D. A. et al., J. Biol. Chem., 264, 10200 (1989)] and AT III Denver wherein Ser at the 394-position has mutated into Leu [see Stephens, A. W. et al., J. Biol. Chem., 262, 1044 (1987)], abnormal AT III molecules each have lost antiprotease activity and patients of these anomalies suffer from thrombotic disorders.
On the other hand, studies on congenital AT III molecule anomalies and results of chemical modification of amino acid residues have revealed amino acids directly relating to the heparin-binding site, namely, binding to heparin. Regarding the molecular anomaly, there have been reported AT III Rouen III wherein Ile at the 7-position has mutated into Asn [see Brennan, S. O. et al., FEBS Lett., 237, 118 (1988)], AT III Rouen IV wherein Arg at the 24-position has mutated into Cys [see Borg, J. Y. et al., FEBS Lett., 266, 163 (1990)], AT III Basel wherein Pro at the 41-position has mutated into Leu [see Chang, J. Y. and Tran, T. H., J. Biol. Chem., 261, 1174 (1986)], AT III Toyama wherein Arg at the 47-position has mutated into Cys [see Koide, T. et al., Proc. Natl. Acad. Sci. USA, 81, 289 (1984)] and AT III Geneva wherein Arg at the 129-position has mutated into Gln [see Gandrille, S. et al., J. Biol. Chem., 265, 18997 (1990)]. Each of these abnormal AT IIIs has a lowered heparin affinity and cannot exert normal physiological functions, thus causing thrombotic disorders. Further, the results of experiments on chemical modification of amino acids suggest that amino acids including Trp at the 49-position, Lys at the 114-position, Lys at the 125 -position, Arg at the 129-position, Lys at the 136-position and Arg at the 145-position might directly relate to binding to heparin [see Blackburn, M. N. et al., J. Biol. Chem., 259, 939 (1984); Peterson, C. et al., J. Biol. Chem., 262, 8061 (1987); Sun, X. J. and Chang, J. Y., Biochemistry, 29, 8957 (1990)].
Based on these findings, attempts have been made to improve AT III through substitution of an amino acid(s) of AT III. For example, Zettlemeissl et al. have disclosed a method for producing an AT III mutant having improved properties relating to heparin binding/heparin activation by mutating an amino acid(s) at the glycosylation site in AT III and another method for producing an AT III mutant having modified enzyme specificities by mutating an amino acid(s) at the reactive site (European Patent Publication-A No. 384122). Further, Dijkema et al. has reported a method for producing an AT III mutant having a modified antithrombin/antiXa activity by mutating an amino acid(s) at the reactive site (International Publication No. WO 91/00291).
However there has not been found any human AT III mutant which is satisfactory from the clinical viewpoint. It is, therefore, urgently required to construct a human AT III mutant having an elevated activity of inhibiting thrombin or factor Xa in the absence of heparin.
It is an object of the present invention to provide novel human AT III mutants having a high antithrombin activity even in the absence of heparin. It is another object of the present invention to provide a method for mass producing said human AT III mutants by the recombinant DNA technology.